Peptides which enhance transport across tissues and methods of identifying and using the same

ABSTRACT

A method of identifying a peptide which permits or facilitates the transport of an active agent through a human or animal tissue. A predetermined amount of phage from a random phage library or a preselected phage library is administered in vivo or in situ to a site in an animal, such as into the gastro-intestinal tract. At a predetermined time, the phage which is transported across a tissue barrier is harvested at a harvesting site, such as in portal or systemic blood or brain tissue, which is separated from the site of administration by the tissue barrier to select transported phage. This transported phage is amplified in a host. This cycle of events is repeated (using the transported phage produced in the most recent cycle) a predetermined number of times to obtain a selected phage library containing phage which can be transported from the site of administration to the harvesting site. The identity of at least one peptide coded by phage in the selected phage library is determined to identify a peptide which permits or facilitates the transport of an active agent through a human or animal tissue.

This application is a divisional of U.S. application Ser. No. 08/857,046(filed on May 15, 1997 and now U.S. Pat. No. 6,361,938 which issued onMar. 26, 2002) which is a continuation-in-part of U.S. application Ser.No. 08/746,411 (filed on Nov. 8, 1996 and now U.S. Pat. No. 6,117,632which issued on Sep. 12, 2000).

TECHNICAL FIELD

This invention relates to the identification of peptide sequences whichpermit or facilitate the transport of drugs, macromolecules, orparticles, such as biodegradable nano- and microparticles, through humanor animal tissues. In particular, this invention relates to the use ofphage display libraries in a screening assay in order to determine theidentity of peptides sequences which enhance the delivery of thebacteriophage through tissue, such as epithelial cells lining thelumenal side of the gastro-intestinal tract (GIT).

BACKGROUND ART

The epithelial cells lining the lumenal side of the GIT are a majorbarrier to drug delivery following oral administration. However, thereare four recognized transport pathways which can be exploited tofacilitate drug delivery and transport: the transcellular, paracellular,carrier-mediated and transcytotic transport pathways. The ability of aconventional drug, peptide, protein, macromolecule or nano- ormicroparticulate system to “interact” with one of these transportpathways may result in increased delivery of that drug or particle fromthe GIT to the underlying circulation.

In the case of the receptor-mediated, carrier-mediated or transcytotictransport pathways, some of the “uptake” signals have been identified.These signals include, inter alia, folic acid, which interacts with thefolate receptor, mannose and cetylmannoside, which interact with themannose receptor, and cobalamin, which interacts with Intrinsic Factor.In addition, leucine- and tyrosine-based peptide sorting motifs orinternalization sequences exist, such as YSKV, FPHL, YRGV, YQTI, TEQF,TEVM, TSAF, YTRF, which facilitate uptake or targeting of proteins fromthe plasma membrane to endosomes. Phage display libraries can bescreened using specific membrane receptors or binding sites to identifypeptides that bind specifically to the receptor or binding site. Theability of certain motifs or domains of peptides or proteins to interactwith specific membrane receptors, followed by cellular uptake of theprotein:receptor complex may point towards the potential application ofsuch motifs in facilitating the delivery of drugs. However, theidentification of peptides or peptide motifs by their ability tointeract with specific receptor sites or carrier sites, such as sitesexpressed on the apical side of the epithelial sites of the GIT, may notbe able to determine, or may not be the most effective way to determine,the identity of peptides capable of enhancing the transport of an activeagent, especially a drug-loaded nano- or microparticle, through tissuessuch as epithelial lining.

Non-receptor-based assays to discover particular ligands have also beenused. For instance, a strategy for identifying peptides that altercellular function by scanning whole cells with phage display librariesis disclosed in Fong et al., Drug Development Research 33:64-70 (1994).However, because whole cells, rather than intact tissue or polarizedcell cultures, are used for screening phage display libraries, thisprocedure does not provide information regarding sequences whose primaryfunction includes affecting transport across polarized cell layers.

Additionally, Stevenson et al., Pharmaceutical Res. 12(9), S94 (1995)discloses the use of Caco-2 monolayers to screen a synthetic tripeptidecombinatorial library for information relating to the permeability ofdi- and tri-peptides. While useful, this technique does not assess theability of the disclosed di- and tri-peptides to enhance delivery of adrug, especially a drug-loaded nano-or microparticle formulation.

Thus, there exists a need for a method of determining peptide sequencesthat are particularly effective in transporting drugs, includingdrug-loaded nano- and microparticles, across a human or animal tissuebarrier.

DISCLOSURE OF THE INVENTION

The invention provides a method of identifying a peptide which permitsor facilitates the transport of an active agent through a human oranimal tissue. A predetermined amount of phage from a random phagelibrary or preselected phage library is plated unto or brought intocontact with a first side, preferably the apical side, of a tissuesample, either in vitro, in vivo or in situ, or polarized tissue cellculture. At a predetermined time, the phage which is transported to asecond side of the tissue opposite the first side, preferably thebasolateral side, is harvested to select transported phages. Thetransported phages are amplified in a host and this cycle of events isrepeated (using the transported phages produced in the most recentcycle) a predetermined number of times, such as from zero to six times,to obtain a selected phage library containing phage which can betransported from the first side to the second side. Lastly, the sequenceof at least one random peptide coded by phage in the selected phagelibrary is determined in order to identify a peptide which permits orfacilitates the transport of an active agent through a human or animaltissue. The transported phage can be viewed as a combination of atransporter peptide (the at least one random peptide coded by the phage)associated with an active agent payload (the phage) in which thetransporter peptide facilitates the transport of the active agentthrough the tissue. Thus, the random peptides coded by phage in theselected phage library are predictively capable of facilitatingtransport of other active agents, such as dug encapsulated nano- and/ormicroparticles, through the particular tissue.

Preferably, the tissue sample derives from the duodenum, jejunum, ileum,ascending colon, transverse colon, descending colon, pelvic colon,vascular endothelium cells which line the vascular system, vascularendothelial cells which form the blood brain barrier, alveolar cells,liver, kidney, bone marrow, retinal cells of the eye or neuronal tissue.The tissue sample can be either in vitro or in vivo. More preferably,the tissue sample comprises epithelial cells lining the lumenal side ofthe GIT, such as isolated rat colon or small intestine segments orepithelial cells lining the lumenal side of the GIT found in an open orclosed loop animal model system. Other preferred tissue samples areheart, spleen, pancrease, thymus and brain tissue.

Preferably, the polarized tissue cell culture sample is cultured fromGIT epithelial cells, alveolar cells, endothelial cells of theblood-brain barrier, or vascular smooth muscle cells. More preferably,the polarized tissue cell culture sample is a polarized Caco-2 cellculture or a polarized T-84 cell culture.

Preferably the random phage library or selected phage library is broughtinto contact with a tissue barrier in vivo or in situ in an animal. Thephage is administered to a site in the animal and is harvested at a sitewhich is separated from the site of administration by a tissue barrier.Preferable harvesting sites include portal blood, systemic blood, braintissue, liver tissue, kidney tissue, bone marrow tissue, heart tissue,spleen tissue, pancreas tissue, thymus tissue, spinal tissue, neuronaltissue, retinal eye tissue, alveolar tissue, vascular smooth muscletissue, tissue in the vascular endothelium of the blood brain barrier,tissue in the vascular endotheliumn which lines the vascular system,pelvic colon tissue, desending colon tissue, transverse colon tissue,ascending colon tissue, ilium tissue, jejunum tissue, duodenum tissue orcombinations thereof.

Preferably, the active agent is a drug or a nano- or microparticle. Morepreferably, the active agent is a drug encapsulated or drug loaded nano-or microparticle, such as a biodegradable nano- or microparticle, inwhich the peptide is physically adsorbed or coated or covalently bonded,such as directly linked or linked via a linking moiety, onto the surfaceof the nano- or microparticle. Alternatively, the peptide can form thenano- or microparticle itself or can be directly conjugated to theactive agent. Such conjugations include fusion proteins in which a DNAsequence coding for the peptide is fused in-frame to the gene or cDNAcoding for a therapeutic peptide or protein, such that the modified genecodes for a recombinant fusion protein in which the “targeting” peptideis fused to the therapeutic peptide or protein and where the “targeting”peptide increases the absoption of the fusion protein from the GIT.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the phage yield (% phage transported from the apical tobasolateral medium) in the basolateral medium of polarized Caco-2 cellsgrown on snapwells at cycles 1, 2, 3, and 4 of panning of the X30 phagedisplay library. For each cycle, the basolateral medium was sampled both1 hour and 24 hours post addition of phage to the apical medium;

FIG. 2 shows the relative binding to fixed Caco-2 cells of 100 differentphage isolates from the X30 phage display library that were obtainedfrom the basolateral medium at completion of cycle 4 (transport fromapical to the basolateral medium) panning of the X30 phage displaylibrary on Caco-2 snapwells;

FIG. 3 shows the binding of the negative control phage M13mp18 and thetop ten binders, clones 32, 34, 39, 40, 53, 80, 84, 97, 98 and 100,[each at neat, 1:25 and 1:100 dilutions] obtained from the X30 libraryfollowing cycle 4 selection on Caco-2 snapwells to fixed Caco-2 cells.For reference, the ELISA absorbance reading obtained with fixed Caco-2cells which were not treated with phage is included;

FIG. 4 shows the binding of the negative control phage M13mp 18 and thetop ten binders, clones 32, 34, 39, 40, 53, 80, 84, 97, 98 and 100,[each at neat, 1:25 and 1:100 dilutions] obtained from the X30 libraryfollowing cycle 4 selection on Caco-2 snapwells to fixed Caco-2 cells,but where the background absorbance reading obtained from the fixedCaco-2 cells only, to which no phage was added, has been subtracted; and

FIG. 5 is a graphical representation of the binding of the phage clones39, 97 and 100 and the negative control phage M13mp18, using either neatphage samples or the same phage diluted 1:25 and 1:100, to fixed Caco-2cells.

MODES FOR CARRYING OUT THE INVENTION

Surprisingly, this invention discloses a method of identifying peptidesthat are capable of facilitating the delivery or transport of an activeagent such as a drug across human or animal tissues, including withoutlimitation GIT epithelial layers, alveolar cells, endothelial cells ofthe blood-brain barrier, vascular smooth muscle cells, vascularendothelial cells, renal epithelial cells, M cells of the Peyers Patch,and hepatocytes. Furthermore, delivery systems, e.g., nanoparticles,microparticles, liposomes, micelles, could be coated externally with, belinked to or be comprised of these “homing” peptides to permit targeteddelivery of encapsulated drugs across particular tissues. In addition,fusion proteins can be synthesized, either in vivo or in vitro, wherebythe peptide is fused in-frame to a therapeutic peptide or protein activeagent such that the peptide enhances the delivery or transport of thetherapeutic peptide or protein across the tissue.

As used herein, the term human or animal “tissue” includes, withoutlimitation, the duodenum, jejunum, ileum, ascending colon, transversecolon, descending colon, pelvic colon, the vascular endothelium whichline the vascular system, the vascular endothelial cells which form theblood brain barrier, vascular smooth muscle, alveolar, liver, kidney,bone marrow, heart, spleen, pancreas, thymus, brain, spinal, neuronaland retinal eye tissue.

As used herein, the term “polarized tissue cell culture” refers to cellscultured so as to form polarized cell layers including, withoutlimitation, cell cultures derived from GIT epithelial cells, alveolarcells, endothelial cells of the blood-brain barrier, or vascular smoothmuscle cells or any other cell type which upon tissue culturing becomespolarized or adopts morphological characteristics or (topological)structures or appendages specific to that cell type in vivo.

As used herein, the term “active agent” includes, without limitation,any drug or antigen or any drug- or antigen-loaded or drug- orantigen-encapsulated nanoparticle, microparticle, liposome, or micellarformulation capable of eliciting a biological response in a human oranimal. Examples of drug- or antigen-loaded or drug- orantigen-encapsulated formulations include those in which the activeagent is encapsulated or loaded into nano- or microparticles, such asbiodegradable nano- or microparticles, and which have the peptideadsorbed, coated or covalently bonded, such as directly linked or linkedvia a linking moiety, onto the surface of the nanny or microparticle.Additionally, the peptide can form the nano- or microparticle itself orthe peptide can be covalently attached to the polymer or polymers usedin the production of the biodegradable nano- or microparticles ordrug-loaded or drug-encapsulated nano- or microparticles or the peptidecan be directly conjugated to the active agent. Such conjugations toactive agents include fusion proteins in which a DNA sequence coding forthe peptide is fused in-frame to the gene or cDNA coding for atherapeutic peptide or protein such that the modified gene codes for arecombinant fusion protein.

As used herein, the term “drug” includes, without limitation, anypharmaceutically active agent. Representative drugs include, but are notlimited to, peptides or proteins, hormones, analgesics, anti-migraineagents, anti-coagulant agents, anti-emetic agents, cardiovascularagents, anti-hypertensive agents, narcotic antagonists, chelatingagents, anti-anginal agents, chemotherapy agents, sedatives,anti-neoplastics, prostaglandins and antidiuretic agents. Typical drugsinclude peptides, proteins or hormones such as insulin, calcitonin,calcitonin gene regulating protein, atrial natriuretic protein, colonystimulating factor, betaseron, erythropoietin (EPO), interferons such asα, β or γ interferon, somatropin, somatotropin, somatostatin,insulin-like growth factor (somatomedins), luteinizing hormone releasinghormone (LHRH), tissue plasminogen activator (TPA), growth hormonereleasing hormone (GHRH), oxytocin, estradiol, growth hormones,leuprolide acetate, factor VIII, interleukins such as interleukin-2, andanalogues thereof; analgesics such as fentanyl, sufentanil, butorphanol,buprenorphine, levorphanol, morphine, hydromorphone, hydrocodone,oxymorphone, methadone, lidocaine, bupivacaine, diclofenac, naproxen,paverin, and analogues thereof; anti-migraine agents such assumatriptan, ergot alkaloids, and analogues thereof; anti-coagulantagents such as heparin, hirudin, and analogues thereof; anti-emeticagents such as scopolamine, ondansetron, domperidone, metoclopramide,and analogues thereof; cardiovascular agents, anti-hypertensive agentsand vasodilators such as diltiazem, clonidine, nifedipine, verapamil,isosorbide-5-mononitrate, organic nitrates, agents used in treatment ofheart disorders, and analogues thereof; sedatives such asbenzodiazepines, phenothiozines, and analogues thereof; narcoticantagonists such as naltrexone, naloxone, and analogues thereof;chelating agents such as deferoxamine, and analogues thereof;antidiuretic agents such as desmopressin, vasopressin, and analoguesthereof; anti-anginal agents such as nitroglycerine, and analoguesthereof; anti-neoplastics such as 5-fluorouracil, bleomycin, andanalogues thereof; prostaglandins and analogues thereof; andchemotherapy agents such as vincristine, and analogues thereof.Representative drugs also include antisense oligonucleotides, genes,gene correcting hybrid oligonucleotides, ribozymes, aptamericoligonucleotides, triple-helix forming oligonucleotides, inhibitors ofsignal transduction pathways, tyrosine kinase inhibitors and DNAmodifying agents. As used herein, the term “drug” also includes, withoutlimitation, systems for gene delivery and gene therapeutics, includingviral systems for gene delivery such as adenovirus, adeono-associatedvirus, retroviruses, herpes simplex virus, sindbus virus, liposomes,cationic lipids, dendrimers, imaging agents and enzymes.

As used herein, the term “preselected phage library” refers to libraryconsisting of a subpopulation of a phage display library. Thissubpopulation is formed by initially screening against either a targetmolecule, such as a protein, receptor, enzyme, ion channel, kinase,growth factor or growth factor receptor so as to permit the selection ofa subpopulation of phages which specifically bind to the targetmolecule. Alternatively, the subpopulation can be formed by screeningagainst a target cell or cell type or tissue type, gastrointestinaltrack, blood brain barrier or other tissue or tissue barrier so as topermit the selection of a subpopulation of phages which either bindspecifically to the target cell or target cell type or target tissue ortarget tissue barrier, or which binds to and/or is transported across(or between) the target cell, target cell type or target tissue ortarget tissue barrier either in situ or in vivo. This preselected phagelibrary or subpopulation of selected phages can also be rescreenedagainst the target molecule or cell or tissue, permitting the furtherselection of a subpopulation of phages which bind to the target moleculeor target cell, target tissue or target tissue barrier or which bind toand/or is transported across the target cell, target tissue or targettissue barrier either in situ or in vivo. Such rescreening can berepeated from zero to 30 times with each successive “pre-selected phagelibrary,” generating additional pre-selected phage libraries.

As used herein, the phrase “human or animal tissue” refers to animaltissue explicitly including human tissue.

It has previously been shown that the NH₂-terminal amino acid sequenceof the absorption proteins pIII and pVIII coded by Escherichia colifilementous bacteriophage phage such as fd, can be modified byrecombinant DNA technology to include a library of random peptidesequences of defined length (Cwirla et al., Proc. Natl. Acad. Sci. USA487:6378-6382 (1990)). Thus, a DNA library of modified phage fdsequences, coding for variable pIII or pVIII proteins can be constructedand propagated in E. coli.

This invention discloses the use of phage display libraries such asthese in a random screening approach or a preselected phage library orsubpopulation from a phage display library in a preselected screeningapproach in order to determine the identity of peptide sequences whichenhance the delivery of the bacteriophage from either the apical tobasolateral side or the basolateral to apical side of either culturedmodel systems or ins vitro, in situ or in vivo tissue samples. Peptidesthat enhance the delivery from the apical to basolateral side (e.g., gutside to blood side) can be used to enhance the delivery of active agentsin that direction. The converse holds for peptides that enhance thedelivery from the basolateral to the apical side. For instance, platingon the basolateral side might determine peptides useful for raising amucosal immune response to an antigen administered IV, subcutaneously,transdermally or by the opthalmic route.

The size of the random peptide sequences coded by the libraries can beof any size. The libraries can be designed to code for linear peptides.Alternatively, the libraries can be so designed to contain cysteineresidues at two or more fixed positions and thus code for cyclicpeptides. As discussed further below, a preferred bacteriophage fd(e.g., from libraries L3.6, L3.15, L8.15) is a filamentous phage havingdimensions of approximately 7 nm by 500-900 nm. On its surface, thephage expresses primarily two different proteins, the gene III protein,of which there are 3-5 copies per phage particle, and the gene VIIIprotein, of which there are approximately 2,500 copies. In the phagedisplay system, the genes coding for either gene III or gene VIII havebeen modified to code for and express random peptide sequences of aparticular length, such as 6-mer, 15-mer and 30-mer. In addition,multiple copies of a DNA insert coding, for example, for a random 15-mersequence can facilitate the production of random peptide sequenceslonger than 15-mer. Each library represents between 10⁸ and 10⁹ or morerandom peptide sequences. As such, the phage library can simulate ananoparticle mixture in which the nanoparticles are coated withdifferent peptides of a specified length.

During the construction of phage display libraries it is possible thatmore than one DNA insert (or partial DNA inserts which may arise due toclevage at internal restriction sites in the DNA library or DNA insert)can be cloned into the cloning sites in gene III or gene VIII, resultingin multiple DNA inserts in the resulting vector clone. Such clonescontaining multiple DNA inserts, or derivatives thereof, have thecapacity to code for longer than expected peptides, due to the presenceof the multiple DNA inserts, provided the DNA inserts are in-frame withrespect to the gene III or gene VIII reading frame and/or provided theclones contain internal DNA sequences which are prone to or suseptibleto the process of ribosomal frameshifting during translation in vivo,which in turn can restore the reading frame of the DNA insert withrespect to the translational reading frame of gene III or gene VIII,and/or provided the mRNA coded by the DNA insert is in-frame with geneIII or gene VIII and does not contain internal translational stop ortranslational termination codons, and/or provided any internaltranslational stop or termination codon(s) can be read as a readingcodon(s) by a translational suppressor molecule in vivo, such as the TAGcodon which is decoded by the SupE suppressor in E. coli as a GLN codon.

The peptides coded by triple (or multiple) DNA inserts have the capacityto code for longer and/or more diverse peptides. Such longer peptideshave a greater capacity to adopt secondary and tertiary structures asopposed to shorter peptides, such as a 15-mer peptide. This capacity ofpeptides to adopt defined secondary and/or tertiary structures coded bythose phages containing multiple or triple DNA inserts may in-turnaccount for the selection of these types of phages from random phagedisplay libraries during selection or panning procedures.

Different transport mechanisms operate in epithelial cells. Sometransport mechanisms are carrier mediated, whereby a carrier or receptorwill bind to a ligand and transport the bound ligand into or through theepithelial cell. Other transport systems operate by transcytosis,whereby a carrier or receptor site will bind a ligand, the carrier:ligand complex is internalized by endocytosis and thus delivers a ligand(or drug) into or through the cell. This invention allows for thediscovery of certain peptide sequences that bind to such active carrieror transcytotic transport systems to facilitate drug delivery. However,rather than focusing on one receptor/carrier system, the inventiondiscloses the use of a blind or random or preselected screening approachin order to identify peptide sequences that interact with undefined orunknown receptor/carrier sites in tissues, such as epithelial cells, andfacilitates the delivery of bacteriophage from the apical to basolateralside of polarized cell cultures or model tissue systems. Because thesepeptide sequences can facilitate the delivery of a bacteriophage, theyare likely to be useful in the transport of drugs and particulatesystems, especially the transport of drug loaded or encapsulated nano-and microparticulate systems when coated onto the surface of the same orfusion proteins whereby the peptide is fused to a therapeutic peptide orprotein. In addition, this invention allows for the discovery of certainpeptide sequences that recognize transcellular or paracellular transportroutes or mechnisms in cultured cells or tissues and so facilitate drugdelivery by these transport pathways.

In brief, the screening approach in the in vitro context includescontacting a predetermined amount of phage from a random phage libraryor a preselected phage library with a first side of a human or animaltissue sample or polarized tissue cell culture, harvesting phage whichis transported to the opposite side of the tissue sample or culture toselect transported phage, amplifying the transported phage in a host andidentifying at least one random peptide coded by a transported phage toidentify a peptide which permits or facilitates the transport of anactive agent through a human or animal tissue. If desired, thecontacting, harvesting and amplifying steps can be repeated apredetermined number of times using the transported phage obtained inthe previous cycle. For instance, using polarized tissue cell culturesamples such as Caco-2 cells or T-84 cells or tissue extracts such asisolated rat colon segments, phage can be plated to the apical side ofthe cultured cells or tissue segments. Subsequently, at any desiredtimepoint but usually from 1 hour to 24 hours, the basolateral medium isharvested aseptically and used to reinfect a host, such as male E. colicoding for the F¹ Factor, to produce progeny. The selected phage fromcycle one can be applied to the apical side of the cultured cells ortissue segment and again the phage in the basolateral medium iscollected, titered and amplified. Repetition of this cycle allows forenrichment of phage capable of being transported from the apical tobasolateral side and thus, the % yield of phage appearing in thebasolateral medium increases as the number of cycles increase. Afterrepeating this cycle from 0 to 30 times, preferably 3 to 20 times, theDNA sequence coding for the NH₂-terminal region of the pIII or pVIIIprotein of the purified, selected, amplified phage(s) is determined topermit deduction of the amino acid sequence of the modified phage(s)which confers the advantage of transport from the apical to basolateralside of the cultured or tissue system.

Similar to the in vitro screening approach given above, the screeningapproach in the in vivo context includes contacting a predeterminedamount of phage from a random phage library or a preselected phagelibrary with a first side of a tissue barrier in vivo, harvesting phagewhich is transported to the opposite side of the tissue barrier toselect transported phage, amplifying the transported phage in a host andidentifying at least one random peptide coded by a transported phage toidentify a peptide which permits or facilitates the transport of anactive agent through a human or animal tissue. If desired, thecontacting, harvesting and amplifying steps can be repeated apredetermined number of times using the transported phage obtained inthe previous cycle.

For instance, the phage display library can be purified such as byeither polyethylene glycol precipitations or sucrose density or CsCldensity centrifugations. The purified library can then be resuspended,such as in TBS or PBS buffer, and introduced onto one side of a tissuebarrier, such as injected into the duodenum, jejunum, ileum, colon orother in vivo animal site using, for instance, a closed loop model oropen loop model. Following introduction of the library to one side of atissue barrier, samples of bodily fluids or body tissue located acrossthe tissue barrier, such as samples of the portal circulation, systemiccirculation brain tissue, heart tissue, kidney tissue spleen tissuepancreas tissue, ileal tissue and/or duodenal tissue, are withdrawn atpredetermined time points, such as 0 to 90 minutes and/or 2 to 6 hoursor more.

For instance, an aliquot of the withdrawn bodily fluid sample (e.g.,blood) is used to directly infect a host, such as E. coli, in order toconfirm the presence of phage. The remaining sample is incubated, suchas overnight incubation with E. coli at 37° C. with shaking.Additionally or alternatively, harvested body tissue (e.g., braintissue) can be cut into small pieces, resuspended in steile PBScontaining a cocktail of protease inhibitors and homogenized. Thishomegenate can be incubated, such as overnight with E. coli at 37° withshaking. The amplified phage present in either culture can be sequencedindividually to determine the identity of peptides coded by the phageor, if further enrichment is desired, can be PEG precipitated,resuspended in PBS, and can be either further PEG-precipitated or useddirectly for administration to another animal closed or open GIT loopmodel system followed by collection of bodily fluid or body tissue andsubsequent amplification of the phage transported into such circulationsystems. In this manner, administration of the phage display librarywith, if desired, repeat administration of the amplified phage to theGIT of the animal permits the selection of phage which are transportedfrom the GIT to the portal and/or systemic circulation and/or varioustissues sites of the animal.

If desired, following administration of the phage display library to thetissue barrier (e.g., GIT) of the animal model, the corresponding regionof the tissue barrier can be recovered at the end of the proceduresgiven above. This recovered tissue can be washed repeatedly in suitablebuffers, such as PBS containing protease inhibitors and homogenized,such as in PBS containing protease inhibitors. The homogenate can beused to infect a host, such as E. coli, thus permitting amplification ofphages which bind tightly to the tissue barrier (e.g., intestinaltissue). Alternatively, the recovered tissue can be homogenized insuitable PBS buffers, washed repeatedly and the phage present in thefinal tissue homogenate can be amplified in E. coli. This approachpermits amplification (and subsequent identification of the associatedpeptides) of phages which either bind tightly to the tissue barrier(e.g., intestinal tissue) or which are internalized by the cells of thetissue barrier (e.g., epithelial cells of the intestinal tissue). Thisselection approach of phage which bind to tissues or which areinternalized by tissues can be repeated.

Subsequently, the corresponding peptide sequences coded by the selectedphages, obtained by the procedures above and identified following DNAsequencing of the appropriate gene III or gene VIII genes of the phage,are synthesized. The binding and transport of the synthetic peptideitself across the model cell culture or isolated tissue system (such ascolonic) permits direct assessment of the transport characteristics ofeach individual peptide. In addition, fusion of the selected peptide(s)sequences with other peptides or proteins permits direct assessment ofthe transport of such chimeric proteins or peptides across the modelsystems. Such chimeric proteins or peptides can be synthesized either invitro or by conventional recombinant technology techniques whereby thecDNA coding for the transporting peptide and the cDNA coding for thedrug peptide or protein are ligated together in-frame and are clonedinto an expression vector which in turn will permit expression in thedesired host, be it prokaryotic cells or eukaryotic cells or transgenicanimals or transgenic plants. For instance, the cDNAs coding for themodified NH₂-terminal region of the pIII proteins can be subcloned intothe genes or cDNAs coding for selected protein molecules (e.g.,calcitonin, insulin, interferons, interleukines, cytokines, EPO, colonystimulating factors etc.) and these modified genes or cDNAs can beexpressed in E. coli or suitable mammalian cells or transgenic animalsor transgenic plants. The expressed recombinant proteins can be purifiedand their transcellular, carrier-mediated, transcytotic and/orparacellular transport across human or animal tissue can be verified. Inaddition, the transporting peptides can be used to coat the surface ofnanoparticulate or microparticulate drug delivery vehicles. Suchcoatings can be performed by either direct adsorption of the peptide tothe surface of the particulate system or alternatively by covalentcoupling of the peptide to the surface of the particulate system, eitherdirectly or via a linking moiety or by covalent coupling of the peptideto the polymers used in the production of nanoparticulate ormicroparticulate drug delivery vehicles, followed by the utilization ofsuch peptide:polymer conjugates in the production of nanoparticulate ormicroparticulate drug delivery vehicles.

Description and Preparation of Phage Display Libraries

Three phage display libraries, identified herein as L3.6, L3.15 andL8.15, were obtained from Prof. George P. Smith at the University ofMissouri-Columbia. Each library is in the vector fUSE5, which wasderived from the parent vector “fd-tet”. In the library L3.6, random6-mer libraries are expressed by the gene III of the fd bacteriophageand are displayed on all 5 copies of the resulting protein pIIIproteins. The number of transductant clones amplified is 3.7×10¹¹ andthe size of phage DNA is 9225 bases. In the library L3.15, random 15-merlibraries are expressed by the gene III of the fd bacteriophage and aredisplayed on all 5 copies of the resulting protein pIII proteins. Thenumber of transductant clones amplified (primary amplification) is3.2×10¹¹; (secondary amplification) is 12.1×10¹² and the size of phageDNA is 9252 bases. In the library L8.15, the vector has two genes VIIIin the same genome, one of which is wild type and the other of whichdisplays the foreign residues. The random 15-mer libraries are expressedby one of the two genes VIII of the fd bacteriophage and are displayedon up to approximately 300 copies of the resulting recombinant proteinpVIII proteins. This vector is called f88-4, in which the foreign 15-meris displayed on up to approximately 300 copies of protein pVIII. Thenumber of transductant clones amplified is 2.2×10¹² and the size ofphage DNA is 9273 bases.

A 30-mer phage display library, X30, was obtained from Dr. Jamie S.Scott of Simon Fraser University. The X30 phage display library codesfor random peptide sequences 30 residues in size. This library wasconstructed in the f88.4 vector, which carries a tetracycline resistancegene and has two pVIII genes: the wild type gene and a synthetic gene.The f88.4 library has variable inserts cloned into the synthetic pVIIIgene of the f88.4 vector.

D38 and DC43 are random phage display libraries in which gene III codesfor random peptides of 38 and 43 residues in size, respectively. Theselibraries are described in McConnell et al, Molecular Diversity1:154-176 (1995), U.S. Ser. No. 310,192 filed Sep. 21, 1994, U.S. Ser.No. 488,161 filed Jun. 7, 1995, and WO 96/09411, which references arehereby incorporated by reference.

A large scale preparation of each of the bacteriophage libraries wasmade in the E. coli host strain K91Kan. A single K91Kan colony wasinnoculated into a sterile 50 ml tube containing 20 ml LB broth (Yeastextract (Gibco)—1 g; Tryptone (Gibco)—2 g; NaCl—1 g; and distilledwater—200 ml) together with kanomycin (final concentration 100 μg/ml)and grown to mid log phase with 200 rpm agitation at 37° C. (OD 0.45 at600 nm). The cells were allowed to incubate with gentle shaking (100rpm, 37° C.) for 5 min to regenerate sheared F pili. The cells werepelleted by centrifugation at 2200 rpm for 10 min at room temperature,the supernatant removed and the cells gently resuspended in 20 ml 80 mMNaCl and shaken gently (100 rpm, 37° C.) for 45 min. The cells werecentrifuged again and the cell pellet was gently resuspended in 1 mlcold NAP buffer (NaCl (5 M stock)—1.6 ml; NH₄H₂PO₄ (0.5 M stock, pH7.0)—10 ml; and distilled water—88.4 ml). The cells were stored at 4° C.and remained injectable for 3-5 days.

The primary libraries were amplified by inoculating two 11 flaskscontaining 100 ml terrific broth with 1 ml of an overnight culture ofK91Kan cells (grown in LB+100 μg/ml kanamycin). This culture wasincubated at 37° C. and 200 rpm until the OD₆₀₀ of a 1:10 dilution was0.2 and then further incubated for 5 min at 37° C. and 200 rpm to allowsheared F pili to regenerate. 10 μl of the primary library was added toeach flask with continued slow shaking for 15 min. Each culture waspoured into a prewarmed 21 flask containing 11 LB+0.22 μg/mltetracycline and shaken at 200 rpm for 35 min. 1 ml of 20 mg/mltetracycline was added and 7 μl samples were removed from each flask.The flasks were replaced in an incubator with continued shakingovernight. 200 μl of various serial dilutions (10⁻⁴, 10⁻⁶, 10⁻⁸, 10⁻¹⁰dilutions) of each culture were spread on LB+40 μg/ml tetracycline and100 μg/ml kanamycin plates and incubated overnight. The colonies werecounted.

Large scale purification of phage was accomplished by dividing theculture evenly between two 500 ml centrifuge tubes and centrifuging at5,000 rpm for 10 min at 4° C. The supernatants were transferred to freshtubes and recentrifuged at 8,000 rpm for 10 min at 4° C. The finalcleared supernatants were poured into fresh tubes and the net volume wasnoted. 0.15 vol PEG/NaCl (PEG 8000—100 g; NaCl—116.9 g; and distilledwater—475 ml) was added and the tubes were mixed gently by inversion(×100 times) and stored on ice for >4 h (or overnight at 4° C.).Following centrifugation at 8,000 rpm for 40 min at 4° C., thesupernatant was decanted, recentrifuged briefly and residual supernatantwas removed by pipetting. 10 ml TBS (Tris HCl (pH 7.5)—0.60 g; NaCl—0.88g; and distilled water—100 ml) was added and the tube was incubated at37° C. and 200 rpm for 30 min to dissolve pellet. The tube wascentrifuged briefly and the solutions from both tubes were transferredto a single Oak Ridge tube, centrifuged at 10-15,000 rpm for 10 min at4° C. and the supernatant was removed to a fresh tube. 0.15 vol PEG/NaClwas added and the phage were allowed to precipitate on ice for 1 h. Theprocedures from the addition of 10 ml TBS were repeated. Into a tared 30ml Beckman polyallomer tube, 4.83 g CsCl was added, the tube retared andthe phage solution was added. TBS was added to a net weight of 10.75 g(total volume 12 ml of a 31% w/w solution of CsCl, density 1.3 g/ml). Aratio of 31:69 w/w ratio is essential. Following centrifugation in theultracentrifuge at 20,000 rpm and 4° C. for 48 h, the tube wasilluminated from the top with a visible light source and identify thephage band:

Phage band—upper band, approximately 5 mm, faint, blue, non-flocculent

PEG—lower band, narrow, stringy, flocculent, opaque, white

The fluid was aspirated off to 2 mm above the phage band and the phageband was withdrawn using a sterile wide aperture transfer pipette andplaced in a 26 ml polycarbonate centrifuge tube. The tube was filled tothe shoulder with TBS, mixed and centrifuged at 50,000 rpm for 4 h at 4°C. in the 60Ti rotor (repeated). The pellet was dissolved in 10 ml TB Sby gentle vortexing and allowed to soften overnight in the cold andrevortexed (repeated). The pellet was then dissolved in TBS (2 ml perliter of original culture) by vortexing, allowed to soften overnight at4° C. and revortexed. The tube was centrifuged briefly to drive solutionto the bottom of the tube and transferred to 1.5 ml microtubes. Sodiumazide (0.02%) can be added and the solution can be heated to 70° C. for20 min to kill residual microorganisms. Following microfuging for 1 minto clear the solution, the supernatant was transferred to sterilemicrotubes and stored at 4° C. 200 μl of a 1:100 dilution was scannedfrom 240-320 ran to determine the concentration of physical particlesand titre 10 μl of a 10⁻⁸ dilution on 10 μl of starved K91Kan cells. 200μl of the infections was spread on LB (+40 μg/ml tetracycline and 100μg/ml kanamycin) plates, incubated at 37° C. for 24 h and counted thenumber of colonies to determine the titre of infectious units in thephage stocks.

Culturing of Caco-2, T-84 Cells

The Caco-2 (ATCC designation: CCL 248; derived from a lung metastasis ofa colon carcinoma in a 72-year old male) and T-84 cells (ATCCdesignation: HTB 37; isolated from a primary colonic tumor in a 72 yearold Caucasian male) were cultured initially in 25 cm² flasks, until theyreached confluency. T84 cells were grown in 1:1 DMEM:Ham's F12 mediumcontaining 2 mM glutamine, 15 mM HEPES, 10% fetal calf serum (FCS), 1%MEM non essential amino acids and 50U ml⁻¹ penicillin and 50 μg mlstreptomycin. Caco-2 cells were grown in DMEM+glutamax-1 containing 10%FCS, 1% MEM non essential amino acids, 50U ml⁻¹ penicillin and 50 μgml⁻¹ streptomycin. All cells were incubated at 37° C. in 95% O₂/5% CO₂.At confluence the cells were used to seed snapwells.

The seeding of snapwells was essentially as follows for T-84 cells (aconcentration of 1×10⁶ cells/1.0 ml of medium is required for each 12 mmsnapwell; a 100% confluent flask of T84 contains approximately 8×10⁶cells and would be sufficient to seed 8 snapwells). The flasks weretrypsinised and cells were carefully resuspended, making sure there areno clumps or air bubbles. 2.6 ml of tissue culture medium is placed inthe bottom of the wells and 0.1 ml on the filter and placed in theincubator for 10 mins at 37° C. 1.5 ml of the cell suspension was addedto each filter, being careful not to let any fall into the bottom of thewell. The filter was placed back in the incubator and checked after 24hrs. The cells were routinely monitored for adequate TER using an EVOMchopstick epithelial voltometer (WPI). In the case of Caco-2 cells, theseeding of Caco-2 cells was essentially the same as for T-84 cellsexcept that they are seeded at 5×10⁵ rather than 1×10⁶ cells/snapwell.

The subsequent maintenance and feeding of the cells on the snapwells wasas follows: when feeding the wells, the medium was removed from thebasolateral side of the snapwell first. The medium was removed from themonolayer with a pipette being careful not to touch the filter and then1 ml of growth medium was place onto the apical side and 2 ml of growthmedium into the basolateral side. Spillages of medium on the sides ofthe plate outside the well were checked for and swabbed with a cottonbud moistened with alcohol if necessary. Following seeding on the snapwells, the cells were fed on a daily basis and were cultured on thesnapwells for between 21-30 days, during which time the cellsspontaneously differentiated and become polarized.

Preparation of Intact Rat Colon Mucosae Tissue

Animals are sacrificed (by carbon monoxide), the abdominal cavity wasopened and the colon was located, removed and washed in 1×Hank'sBalanced Salt Buffer (HBSS; Gibco BRL, Cat # 14065-031). The tubularsegment was cut along the mesenteric border to give a flat square pieceof tissue. The smooth muscle layer was then removed by blunt dissectionto leave an approximate 2.5 cm² patch of epithelium.

The isolated rat colonic mucosae were mounted in Side-by-sideSweetana-Grass (SG) diffusion chambers. The mounted rat colonic mucosaein the S-G chambers were used in the analysis of phage transport fromthe apical to basolateral side of the colonic tissue.

Balancing Side-by-side Chambers

The water bath was allowed to equilibrate to 37° C. The chambers werefilled with HBSS Buffer (see below) and the electrodes are switched on.The input-offset control knob was adjusted to zero. The system wasallowed to equilibrate for approximately 20 minutes, making sure thereadings remain at zero throughout The electrodes were switched off andHBSS solution removed. Filters containing sheets of the rat colonicepithelium were mounted on the apparatus and 10 mls of HBSS Buffer wasadded to each side simultaneously. The tissues were oxygenated with 95%O₂/5% CO₂ and the system was allowed to equilibrate for at least 30minutes. Electrodes were switched on and the knobs set to voltage clampand current. Voltage was adjusted to give a change in current ofapproximately 2-3 μA. The timer was then set to apply a voltage every 8mins and the corresponding deflected current was used to calculate TERby applying the following Ohmic relationship: R=V/I. Recordings werecommenced for at least 10 min before any phage was added.

Enzyme Linked Immuno-sorbent Assay (ELISA) for fd-derived Phage onCaco-2 Cells

Caco-2 cells (100 μl) were grown to confluence in 96 well tissue cultureplates (2×10⁵ cells/well grown for 2 days in growth medium containingDMEM/Glutamax+1% Pen/Strep, 1% MEM & 10% FCS). After two days growth,100 μl of 10% formaldehyde [Formaldehyde (38%) sterile distilled water(1:3 vol)] was added to the confluent Caco-2 cell monolayers followed byincubation for 15 min at room temperature. The contents of themicrotitre wells was emptied by inversion/flicking and the wells werewashed three times with DPBS (Dulbecco's PBS). Each well was filled with200 μl of 0.1% phenylhydrazine-DPBS (0.1% phenylhydrazine in DPBS) andincubated for 1 h at 37° C. Subsequently, the contents of the microtitrewells were emptied by inversion/flicking and the wells were washed threetimes with DPBS. 200 μl of 0.5% BSA in DPBS was added to each wellfollowed by incubation for 1 h at room temperature. Each well was nextwashed three times in 1% BPT (1% BSA, 0.05% Tween 20 in DPBS).

Phage samples (100 μl in 1% BPT) (either neat phage at 10¹⁰ pfu/ml or1:25 or 1:100 dilutions thereof) were added to the wells, followed byincubation at room temperature for 2 h. The contents of the microtitrewells were removed by inversion/flicking and the wells were washed fivetimes in 1% BPT. 100 μl of horse-radish peroxidase (HRP)-anti-M13conjugate (HRP/anti-M13 conjugate: horseradish peroxidase conjugated tosheep anti-M13 IgG; 1:5000 working dilution in 1% BPT; Pharmacia27-9402-01) was added to each well, followed by incubation for 1 h atroom temperature. The contents of the microtitre wells were againremoved by inversion/flicking and the wells were washed five times in 1%BPT. 200 μl of TMB substrate solution (3,3′,5′,5-tetramethylbenzidine;Microwell Peroxidase Substrate System; Kirkegaard & Perry LaboratoriesCN 50-76-00; prepared by mixing equal amounts of TMB PeroxidaseSubstrate A and Peroxidase Solution B in a glass container immediatelybefore use) was added to each well, followed by incubation at roomtemperature for 20-60 min. Thereafter, absorbance readings were read at650 nm on a microtitre plate reader.

Processing of Harvesting Site Tissue

Harvesting site tissue, such as brain, heart, kidney, spleen, liver,pancreas, duodenum or ileum tissue, is collected from animals followingintroduction in vivo of the phage display library at a site separatedfrom the harvesting site by a tissue barrier. The animals are sacrificedat a predetermined time following administration of the library, thetissue is removed and the tissue is either processed immediately orfrozen in liquid nitrogen (stored at −80° C.) for processing at a laterdate. The tissue samples are homogenised in PBS containing proteaseinhibitors and the homogenate is used to infect E. coli, thus permittingamplification of phages that were transported to the harvesting sitetissue.

Processing of Tissue Adjacent the Phage Display Library AdministrationSite

For use in the in vivo embodiment described herein, the phage displaylibrary is purified such as by either PEG precipitation or by sucrose orCsCl density centrifugation. The phage display library is resuspended inPBS (or TBS) buffer and injected into the in vivo animal site, such asduodenum, jejunum, ileum, colon, ascending colon, transverse colon,descending colon, pelvic colon in the closed (or open) animal (rat,rabbit or other species) loop model. Following administration of thephage display library to the gastrointestinal tract of the animal model,and withdrawal of portal and/or systemic blood samples at predeterminedtime points (such as 0 min, 15 min 30 min, 45 min, 60 min up to 6hours), or incubation of the administered phage display library in theclosed (or open) loop model for a predetermined period of time, thecorresponding region of the GIT track exposed to or incubated with thephage display library can be recovered at the end of the experiments.Following repeated washings of the recovered intestinal tissue insuitable buffers such as PBS containing protease inhibitors, the washedtissue is homogenised in PBS containing protease inhibitors and thehomogenate is used to infect E. coli, thus permitting amplification ofphages which can bind tightly to the intestinal tissue. Alternatively,the recovered intestinal tissue can be homogenised in suitable PBSbuffers, washed repeatedly and the phage present in the final tissuehomogenate can be amplified in E. coli. This latter approach alsopermits amplification of phages which either bind tightly to theintestinal tissue or which are internalized by the epithelial cells ofthe intestinal tissue

Selection of Phage with Enhanced Ability to Cross Cellular Barriers

A. Treatment of Tissue Culture Cell Monolayers (Snapwell Models) withPhage Display Populations

In a laminar flow cabinet, 100 μl of phage solution was mixed with 900μl of growth medium without antibiotic (the complete recommended mediumfor each cell line but with no antibiotics added) in a microfuge tube.The experiment was carried out in duplicate and included a controltreatment containing no phage. The TER was measured for each snapwell,noting the age of the cells and the passage number. Only intactmonolayers of recommended age were used which had expected TER. Thebasolateral medium was replaced in the snapwells with medium withoutantibiotic and the apical medium was removed. The phage solutions andcontrol solutions were added to the apical side of the cells and thesnapwell cultures were incubated as normal. At each harvest time point(e.g., 1 h, 5 h, 24 h after application of phage), the medium wasremoved from the basolateral side and stored in a sterile 2 ml screwcaptube at 4° C. At each time that the basolateral medium is removed, themedium was replaced with fresh medium without antibiotic. When theexperiments are finished, the TER was measured and the monolayers weretreated with Vircon disinfectant as per normal.

The phage were titrated by preparing starved cells of E. coli K91Kan andcarrying out serial dilutions of phage in the (growth medium above) inTBS/gelatin. 10 μl of starved cells and 10 μl of serially-diluted phagesolution were mixed in a 1.5 ml microfuge tube. The phage was allowed toinfect for 10 min at room temperature. In general, the followingdilutions are used:

Sample Dilution t = 1 h neat or 10⁻¹ t = 5 h 10⁻¹, 10⁻³ t = 24 h 10⁻¹,10⁻³ Apical/amplified 10⁻⁶, 10⁻⁷, 10⁻⁸1 ml of LB medium containing 0.2 μg ml⁻¹ tetracycline was added to thephage/K91Kan cell mixtures and incubated for 30 min at 37° C. 200 μl ofthe phage/K91Kan cell mixture was spread on LB agar plates containing 40μg ml⁻¹ tetracycline and 100 μg/ml kanomycin and grown overnight at 37°C. For a 10⁻² dilution (10 μl into 990 μl), 200 colonies on a platerepresents 1×10⁷ TU ml⁻¹.

Thus, by estimating the titre of phage which was present in thebasolateral medium and by knowing the number of phage that was appliedto the apical side, an estimate of the % yield of phage transported tothe basolateral medium from the apical side can be made.

Selected phage present in the basolateral growth medium were amplifiedby adding 150 μl of PEG/NaCl per 1 ml of phage solution (pool theharvest from all the three time-points (eg. 3×2 ml=6 ml) in an Oak Ridgetube. The solution is mixed very well by continuously inverting for 2-3min and stored at 4° C. for at least 4 h. The precipitated phage iscentrifuged for 15 min at 10,000 g (8,500 rpm using Beckman JA17 rotor)in a Beckman J2-MC preparative ultracentrifuge. The supernatant wasremoved and recentrifuged as before for 5 min. The pellet wasresuspended in 100 μl of TBS by leaving for 5 min at room temperatureand vortexing (repeat by leaving for 15 min and vortexing again). Thesuspended phage solution was placed in an Oak Ridge tube and 100 μl ofstarved E. coli K91Kan cells were added. The phage/cell solution wasmixed gently and left at room temperature for 30 min. 20 ml of prewarmedLB medium containing tetracycline (0.2 μg ml⁻¹) and kanomycin (100μg/ml) was added and incubated at 200 rpm at 37° C. for 30 min. 10 μl ofstock tetracycline (40 mg ml⁻¹) was added to the medium and the tube wasincubated overnight. The overnight culture was centrifuged for 15 min at3440 g (5,000 rpm using Beckman JA17 rotor) in a Beckman J2-MCpreparative ultracentrifuge. The supernatant was added to a clean(preferably sterile) Oak Ridge tube and centrifuged again for 10 min at13800 g (10,000 rpm). The supernatant was placed in a clean (preferablysterile) Oak Ridge tube containing 3 ml of PEG/NaCl and mixed bycontinuous inversion for 2-3 min. Following storage at 4° C. for atleast 4 h, the tube was centrifuged for 15 min at 13800 g (10,000 rpmusing Beckman JA17 rotor) in a Beckman J2-MC preparativeultracentrifuge. The supernatant was removed and recentrifuged as abovefor 5 min at 10,000 rpm. As much supernatant as possible was removedwith a micropipette and the pellet was resuspended in 1 ml of TBS byleaving for 5 min at room temperature and vortexing. The resuspensionwas left for 15 min and vortexed again. The phage solution wastransferred to a 1.5 ml microfuge tube and vortexed again. The solutionwas centrifuged at 13,000 rpm for 30 s in a microfuge and thesupernatant was transferred to a fresh 1.5 ml microfuge tube containing150 μl PEG/NaCl. The tube was mixed by inverting for 2-3 min and storedat 4° C. for at least 1 h. Subsequently, the tube was centrifuged at13,000 rpm for 10 min in a microfuge and the supernatant was removed andrecentrifuged for 5 min. The pellet was resuspended in 100 μl of TBS byleaving for 5 min at room temperature and vortexing. The resuspensionwas left for 15 min and vortexed again. This resuspension represents thephage selected in cycle 1. One μl should be withdrawn and used fortitration to confirm that approximately 10⁹ TU are present.

The phage solution is now ready for a further round of selection in thecultured T84 and Caco-2 cells, by repeating the steps above using thephage transported into the basolateral medium. Thus, phage selected fromcycle one is now reapplied to the apical side of the Caco-2 or T-84cells growing on Snapwells. In general, in each cycle the same titre ofphage is applied to the apical side of the cells growing on snapwells.At the end of each cycle the titre of phage present in the basolateralmedium at each time point is determined and these transported phage arereamplified and recycled back through the cells. Thus, the % yield ofphage which appear in the basolateral medium increases as the number ofcycles increase. At the end of cycle five, phage have been selectedwhich are preferentially transported from the apical to basolateral sideof the cultured cells, due to the random peptide sequences displayed bythe bacteriophage gene III or gene VIII protein products.

B. Treatment of Intact Rat Colon Mucosae Tissue with Phage DisplayPopulations

Once the rat colonic tissue is set up as described above, approximately1×10¹¹ phage in HBSS buffer were applied to the gut side of the colonictissue, after the electrodes were switched off. Subsequently, atindicated time points, the settings were changed to voltage and amplify,the system was grounded, the medium on both the gut side and blood sideof the colonic tissue were simultaneously removed, and the medium on theblood side was saved at 4° C. The original medium present on the gutside was replaced onto the gut side of the mounted colonic tissue in theS-G chambers. Simultaneously fresh HBSS buffer medium was added to theblood side, and the tissues were oxygenated with 95% O₂/5% CO₂.Electrodes were switched on again and the knobs set to voltage clamp andcurrent. Voltage was adjusted to give a change in current ofapproximately 2-3 μA. The timer was then set to apply a voltage every 8mins and the corresponding deflected current was used to calculate TERby applying the following Ohmic relationship: R=V/I.

The phage post transfer across rat colon was titrat and amplified asfollows (phage samples titred prior to and after amplification). Serialdilutions of phage (2 μl phage+18 μl TBS/gelatin) were performed inmicrotitre plates and 10 μl volumes of the required dilutions weretransferred to 1.5 ml microtubes. 10 μl of starved K91Kan cells wereadded to each microtube, mixed gently and incubated at room temperaturefor 10 min. 990 μl of LB+0.2 μg ml⁻¹ tetracycline were added and themicrotubes were incubated at 37° C. for 30 min. 200 μl of the culturewere spread on LB (40 μg ml⁻¹ tetracycline+100 μg ml⁻¹ kanamycin) agarplates, incubated at 37° C. overnight and the number of colonies werecounted.

The phage was amplified by adding 150 μl of PEG/NaCl to 1 ml of phagesolution (i.e., apical or basolateral HBSS buffer from chambers) in anOak Ridge tube, mixing by inversion (×100) and incubating at 4° C. for 4h. The tube was centrifuged at 10,000 g for 15 min (JA17 rotor, 8,500rpm) and the supernatant was decanted and recentrifuged for 5 ml. Thesupernatant was removed and the pellet was resuspended in 100 pi of TBS(leave at room temperature for 5 min, vortex, leave at room temperaturefor 15 min and revortex). A 5 μl sample was retained for titration. 100μl of starved K91Kan cells were added to 95 μl of phage solution, mixedgently and incubated at room temperature for 30 min. 20 ml of pre-warnedLB+0.2 μg ml⁻¹ tetracycline were added and the tube was incubated at 37°C. and 200 rpm for 30 min. 10 μl of tetracycline (40 mg ml⁻¹ stock) andkanomycin (final concentration of 100 μg/ml) were added and the tube wasincubated overnight at 37° C. and 200 rpm. The tube was then centrifugedfor 15 min at 3440 g (JA17 rotor; 5,000 rpm), the supernatant was addedto a new Oak Ridge tube and recentrifuged at 13,800 g (JA17 rotor;10,000 rpm). The supernatant was transferred to a new Oak Ridge tubecontaining 3 ml of PEG/NaCl, mixed by inversion (×100) and incubated at4° C. for 4 h. The tube was then centrifuged at 13,800 g, thesupernatant decanted and recentrifuged at 13,800 g for 5 min. The pelletwas resuspended in 100 μl of TBS (leave at room temperature for 5 min,vortex, leave at room temperature for 15 min and revortex). The phagesolution was transferred to a microtube containing 150 μl of PEG/NaCl,mixed by inversion (×100) and incubated at 4° C. for 1 h. The tube wasmicrofuged for 1 min, the supernatant removed and remicrofuged. Thesupernatant was removed and the pellet resuspended in 100 μl of TBS(leave at room temperature for 5 min, vortex, leave at room temperaturefor 15 min and revortex). 2 μl of phage for was removed for titrationwhile the rest was stored at 4° C.

The phage solution is now ready for a further round of selection in theS-G mounted rat colonic tissue, by repeating the steps above using thephage transported into the basolateral medium. Thus, phage selected fromcycle one is now reapplied to the apical or gut side of the S-G mountedrat colonic tissue. In general, in each cycle the same titre of phage isapplied to the gut side of the tissue. At the end of each cycle thetitre of phage present in the basolateral medium (blood side) at eachtime point is determined and these transported phage are reamplified andrecycled back through the colonic tissue. Thus, the % yield of phagewhich appear in the basolateral medium increases as the number of cyclesincrease. At the end of cycle five or six we have selected for phagewhich are preferentially transported from the apical or gut side of thecolonic tissue to blood side or basolateral side of the colon tissue,due to the random peptide sequences displayed by the bacteriophage geneIII or gene VIII protein products.

C. Treatment of Animal Tissue Barriers in vivo with Phage DisplayPopulations

The purified phage display library (random or preselected) is diluted to500 μl in PBS buffer and injected into the closed (or open) intestinalloop model (e.g., rat, rabbit or other species). At time 0 and atsuccessive time points after injection, a sample of either the portalcirculation or systemic circulation is withdrawn. An aliquot of thewithdrawn blood can be incubated with E. coli, followed by plating forphage plaques or for transduction units or for colonies where the phagecodes for resistance to antibiotics such as tetracycline. The remainderof the withdrawn blood sample (up to 150 μl) is incubated with 250 μl ofE. coil and 5 ml of LB medium or other suitable growth medium. The E.coli cultures are incubated overnight by incubation at 37° C. on ashaking platform. Blood samples taken at other time points (such as 15min, 30 min, 45 min, 60 min up to 6 hours) are processed in a similarmanner, permitting amplification of phages present in the portal orsystemic circulation in E. coli. at these times. Followingamplification, the amplified phage is recovered by PEG precipitation andresuspended in PBS buffer or TBS buffer. In addition, the titer of theamplified phage, before and after PEG precipitation is determined. Theamplified, PEG precipitated phage is diluted to a known phage titer(generally between 10⁸ and 10¹⁰ phage or plaque forming units per ml)and is injected into the GIT of the animal closed (or open) loop model.Blood samples are collected from portal and for systemic circulation atvarious time points and the phage transported into the blood samples areamplified in E. coli as given above for the first cycle. Subsequently,the phage are PEG precipitated, resuspended, titered, diluted andinjected into the GIT of the animal closed (or open) loop model. Thisprocedure of phage injection followed by collection of portal and/orsystemic blood samples and amplification of phage transported into theseblood samples can be repeated, for example, up to 10 times, to permitthe selection of phages which are preferentially transported from theGIT into the portal and/or systemic circulation.

Additionally or alternatively, at the conclusion of the portal bloodsampling (typically within 60 ml from administration of the phagedisplay library) or systemic blood sampling (typically within 360 minfrom administration of the phage display library) or at other convenientpredetermined times, the animal can be sacrificed and the harvestingsite tissue, such as brain tissue, removed. The tissue can be processedimmediately or frozen in liquid nitrogen (stored at −80° C.) forprocessing at a later date. Following homogenization of the tissue inPBS containing protease inhibitors, serial dilutions (such as neat,10⁻², 10⁻⁴, 10 ⁻⁶ of dilutions) of the tissue homogenate are titered inE. coli. An aliquot (100 μl) of the tissue homgentate is added to 100 μlof E. coli K91Kan starved bacteria, and incubated at 37° C. for 10 minfollowed by addition of 5 ml of LB medium or other suitable growthmedium. The E. coli cultures are incubated overnight at 37° C. andserial dilutions of amplified phage are then titered in E. coli. Asabove, the recovered phage can be administered to other animals,harvesting site tissue can be collected, and the phage transported intothe tissues can be amplified repeatedly, for example, up to 10 times, topermit the selection of phages which are preferentially transported fromthe GIT into the harvesting site tissue.

EXAMPLE 1 % Yield of φ in Caco-2 Cells

Libraries L3.6, L3.15, L8.15 and fUSE2 (control) were screened usingCaco-2 cells according to the procedures given above. The percentageyields per cycle (1 hr, 5 hr, 24 hr and total yield) and the change intransepithelial resistance for the cycles were measured. The TERmeasurements for the Caco-2 cells remained in the range 224-449 Ωcm⁻².The phage yield on the basolateral side of the cell culture is reportedas a percentage of the phage applied to the apical side. Six successivescreening cycles were performed and 1 hr, 5 hr and 24 hr samples of thebasolateral buffer were harvested. The percentage yields of phageobtained per cycle in cycles 1-6 are summarized in Table 1. Usableyields were generally obtained by the 4th cycle.

EXAMPLE 2 % Yield of φ in T-84 Cells

Libraries L3.6, L3.15, L8.15 and fUSE2 (control) were screened usingT-84 cells according to the procedures given above. The percentageyields per cycle (1 hr, 5 hr, 24 hr and total yield) and the change intransepithelial resistance for the cycles were measured. The TERmeasurements for the T-84 cells remained in the range 224-449 Ωcm⁻². Thephage yield on the basolateral side of the cell culture is reported as apercentage of the phage applied to the apical side. Four successivescreening cycles were performed and 1 hr, 5 hr and 24 hr samples of thebasolateral buffer were harvested. The percentage yields of phageobtained per cycle in cycles 14 are summarized in Table 2. Usable yieldswere generally obtained by the 4th cycle.

EXAMPLE 3 % Yield of φ in Isolated Colon Segments

A phage mixture comprising libraries L3.6, L3.15 and L8.15 was screenedusing isolated rat colon according to the procedures given above. Thephage yield on the basolateral side of the tissue sample is reported asa percentage of the phage applied to the apical side. Six successivescreening cycles were performed and four 1 h samples of the basolateralbuffer were harvested. Table 3 reports the % yield of φ in isolatedcolon segments.

TABLE 1 % YIELD 0F φ IN CACO-2 CELLS Time (hours) Round 1 5 24 TotalLibrary L3.6 1   9 × 10⁻¹   9 × 10⁻¹    9 × 10⁻¹ 0.0027 2   5 × 10⁻⁴0.016 0.077 0.0935 3 1.56 × 10⁻⁵ 0.0625 0.14 0.202 4 0.132 0.44 0.03360.6056 5 1.64 × 10⁻⁴ 0.069 1.377 1.45 6 3.88 × 10⁻³ 5.93 × 10⁻⁴  3.04 ×10⁻³ 0.0075 Library 3.15 1  9.5 × 10⁻¹  9.5 × 10⁻⁴  9.5 × 10⁻⁴ 0.00285 2  5 × 10⁻⁴ 20 10 30.0 3  2.5 × 10⁻⁵ 1.35 × 10⁻³ 15 15.0 4 0.207 0.0480.82 1.075 5   2 × 10⁻⁴ 0.21 2.875 3.09 6 1.17 × 10⁻⁵ 19.2 6.4 25.6Library L8.15 1 0.02 0.02 0.02 0.02 2   5 × 10⁻⁴ 0.5 0.018 0.5185 3  1.4× 10⁻³ 0.077 1.57 1.6484 4 2.84 × 10⁻⁴ 5.39 × 10⁻³ 0.14 0.1456 5 2.44 ×10⁻⁴ 0.097 1.805 1.902 6 0.0142 70.5 38 108 Library fUSE2 (control) inCaco-2 cells 1 0.02 0.02 0.02 0.02 2   5 × 10⁻⁴   5 × 10⁻⁴ 0.03 0.031 32.08 × 10⁻⁵ 2.08 × 10⁻⁵ 1.125 × 10⁻³ 0.001145 4   5 × 10⁻⁴   5 × 10⁻⁴   5 × 10⁻⁴ 0.0005 (?) (?) (?) 5 2.34 × 10⁻³ 0.117 0.025 0.14 6 9.3952.5 94 155.89

TABLE 2 % YIELD OF φ IN T-84 CELLS Time (hours) Round 1 5 24 TotalLibrary L3.6 1  3.33 × 10⁻⁶ 1.66 × 10⁻⁶ 2.4 2.4 2  7.9 × 10⁻³ 0.27739.68 39.957 3  9.8 × 10⁻⁵  9.8 × 10⁻⁵ 1.04 1.04 4 0.0274 0.22 1.05 1.30Library L3.15 1  4.08 × 10⁻⁴  5.8 × 10⁻³ 0.016 0.0218 2 0.342 0.054 1.782.176 3(*)  4.3 × 10⁻⁴  4.3 × 10⁻⁴ 2.28 2.28 4 0.00 8.62 6.7 15.32Library L8.15 1  2.7 × 10⁻⁶  2.7 × 10⁻⁶ 2.9 × 10⁻⁴ 0.00029 2  2.6 × 10⁻⁴0.36 13.02 13.38 3  1.06 × 10⁻⁴ 1.06 × 10⁻⁴ 0.57 0.57 4 4.495 × 10⁻³52.9 40.2 93.1 Library fUSE2 (control) in T-84 cells 1    1 × 10⁻³   1 ×10⁻³   1 × 10⁻³ 0.001 2 3  2.35 × 10⁻⁴ 0.046 7.6(*) 7.6(*) 4 4.00 1.4040.634 6.038 5 2.4 × 10⁻⁴/3 × 10⁻⁴

TABLE 3 % YIELD OF φ IN ISOLATED COLON SEGMENTS % yield Cycle Time (h)Chamber A Chamber B 1 1 4.1 × 10⁻⁶ 4.1 × 10⁻⁶ 2 0 8.2 × 10⁻⁶ 3 0 4.1 ×10⁻⁶ 4 0 0 Total: 4.1 × 10⁻⁶ Total: 1.6 × 10⁻⁵ 2 1 2.6 × 10⁻⁶ 2.3 × 10⁻⁶2 0 0 3 0 0 4 0 2.3 × 10⁻⁶ Total: 2.6 × 10⁻⁶ Total: 4.6 × 10⁻⁶ 3 1 1.4 ×10⁻⁴ 2.5 × 10⁻⁴ 2 8.5 × 10⁻⁵ 4.2 × 10⁻⁴ 3 7.5 × 10⁻⁵ 6.4 × 10⁻⁴ 4 7.5 ×10⁻⁵ 6.5 × 10⁻⁴ Total: 3.7 × 10⁻⁴ Total: 2.0 × 10⁻³ 4 1 0 0 2 0 0 3 01.2 × 10⁻⁵ 4 0 0 Total: 0 Total: 1.2 × 10⁻⁵ 5 1 2.3 × 10⁻⁴ 2.1 × 10⁻³ 24.725 0.049 3 1.7 × 10⁻³ 1.6 × 10⁻⁵ 4 0.0675 4.2 × 10⁻⁵ Total: 4.79Total: 0.051 6 1   7 × 10⁻³ 0.024 2 2.8 × 10⁻³ 0.03 3 7.5 × 10⁻³ 0.056 45.6 × 10⁻³ 0.048 Total: 0.023 Total: 0.16

EXAMPLE 4 Identification of Peptide Sequences from Transported Phage inColon Tissue Segments

Thirty-six clones from randomly selected phages from the sixth cycle ofscreening in rat colon segments (as given in Example 3 and Table 3) weresequenced using either the gene VIII DNA sequencing primer ELN71 (SEQ IDNO: 1) or the gene III DNA sequencing primer ELN77a (SEQ ID NO: 17),³⁵S-dATP and the Sequenase version 2.0 DNA sequencing kit (Amersham LifeScience, UK). Progressing from cycle 1 to cycle 6, there is a bias inthe selection of phage with random peptides coded by gene VIII asopposed to gene III, perhaps because the gene III coded peptides arepresent between 3-5 copies/phage particle whereas the synthetic geneVIII coded peptides are present at around 300 copies per phage particle.This higher expression level may provide a valency effect and increasethe possibility of interaction with a receptor site/pathway in thetissue sample.

A number of clones/DNA sequences are present more than once, suggestingsome type of preferential selection. Thus, SEQ ID NO: 2 (a Class of 9clones—25% presence), SEQ ID NO: 3 (a Class of 5 clones—13.9% presence),SEQ ID NO: 4 (a Class of 3—8.3% presence) were determined from this 36clone sample from cycle 6. All of these Classes consist of clones withtriple DNA inserts. Individual isolates are given by SEQ. ID. NO: 5 toSEQ ID NO: 9 (triple DNA inserts) and SEQ ID NO: 10 (single insert).

Based on the recurrent random peptide sequences in these classes, twosynthetic oligonucleotides were constructed and used to screen phagepopulations representing colon screening cycles 1-6 in a series ofoligonucleotide hybridization reactions to determine whether these phageand corresponding peptides were being selected during the screeningprocess. Thus, oligonucleotides ELN93 and ELN94 correspond to a partialcoding region in those phage clones for SEQ ID NO: 2 and SEQ ID NO: 3,respectively. The incidence of reactivity per screening cycle issummarized in Table 4 below. From the data presented in Table 4, itappears that there is a gradual selection of phage which hybridize tooligonucleotide ELN93 and ELN94 progressing from cycle 1 through cycle6. Probe reactivity is expressed as a percentage of the total number ofcolonies screened per phage population. As a control, the unselected,starting libraries (L3.6, L3. 15 and L8.15) were also included.

TABLE 4 HYBRIDIZATION OF PHAGE POPULATIONS (COLON SCREENING CYCLES 1-6AND UNSELECTED LIBRARIES L3.6, L3.15 AND L8.15) WITH OLIGONUCLEOTIDESELN93 AND ELN94 ELN93 ELN94 1 0.4 0.4 2 4.7 0 3 17.4 0 4 26.4 1.25 >20.0 >40.0 6 62.5 >40.0 L3.6 0.8 0 L3.15 0.8 0 L8.15 0.3 0

The phage populations representing Caco-2 screening cycles 1-6 and T-84screening cycles 1-4, as given above in Example 1, Table 1 and Example2, Table 2, respectively, were also assessed for reactivity to theoligonucleotide probes ELN93 and ELN94. The incidence of reactivity perscreening cycle in Caco-2 and T-84 cells is compared to reactivity incolon tissue in Table 5 (ELN93) and Table 6 (ELN94). In these Tables,probe reactivity is expressed as a percentage of the total number ofcolonies screened per phage population. Some reactivity was detected inCaco-2 selected clones using ELN93. The gradual selection of ELN93reactive phage during progression from cycles 1 to 6 observed for phagelibrary L3.15B correlated with the pattern of reactivity previouslyobserved for colon-selected phage although the overall reactivityachieved was substantially lower. ELN94 reactivity was identified inboth Caco-2 and T-84 selected clones. Increasing reactivity from cycles1 to 6 was observed for Caco-2 selected libraries L3.6B, L3.15B andL8.15B as well as the T-84 selected library L3.15A. The reactivity ofthe Caco-2 selected libraries L3.6B and L8.15B at cycle 5 (.33.3% and42.3%, respectively) was remarkably similar to that of colon A selectedphage (46.0%).

Table 5: Hybridization of Phage Populations with Oligonucleotide ELN93

Table 5.a: Caco2 Screening Cycles 1-6, Colon Screening Cycles 1-6 & T-84Screening Cycles 1-6

Table 5.b: Unselected Libraries L3.6, L3.15 & L8.15

TABLE 5.a Caco2 Caco2 Caco2 Caco2 Caco2 Caco2 Colon Colon T-84 T-84 T-84T-84 T-84 T-84 Cycle 3.6A 3.6B 3.15A 3.15B 8.15A 8.15B A B 3.6A 3.6B3.15A 3.15B 8.15A 8.15B 1 0 0.3 0 0 0 0 0.4 NA NA NA NA NA NA NA 2 0 0.30 0.4 0.3 1.0 4.7 NA 0 0 0 0 0 0 3 0 0 0 0.8 0 0 17.4 NA 0 0 0 0 0 0 4 00 0 1.2 0.3 0 26.4 NA 0 0 0 0 0 0 5 0 0 0 7.2 0 4.9 >20.0 NA NA NA NA NANA NA 6 NA NA NA NA NA NA 62.5¹ 0.6 0 0 0 0 0 0 (0.8)² ¹Assay 1 ²Assay 2NA Not assayed

TABLE 5.b Unselected libraries ELN93 L3.6 0.8 L3.15 0.8 L8.15 0.3

Table 6: Hybridization of Phage Populations with Oligonucleotide ELN94

Table 6.a: Caco-2 Screening Cycles 1-6, Colon Screening Cycles 1-6 & T84Screening Cycles 1-6

Table 6.b: Unselected Libraries 13.6, L3.15 & L8.15

TABLE 6.a Caco2 Caco2 Caco2 Caco2 Caco2 Caco2 Colon Colon T-84 T-84 T-84T-84 T-84 T-84 Cycle L3.6A L3.6B 3.15A 3.15B 8.15A 8.15B A B 3.6A 3.6B3.15A 3.15B 8.15A 8.15B 1 0 0 0 0 0 0 0.4 NA NA NA NA NA NA NA 2 0 0.3 00 0 0 0 NA 0 0 0 0.4 0 0.4 3 0 0 0 0 0 1.4 0 NA 0 0 2.8 0 0 2.8 4 0 6.00 1.6 0 (3.8) 12.9 1.2 NA 4.0 0 12.8 4.4 0 0.4 5 3.3 >33.3 3.3 6.0 4.042.3 >40.0 NA NA NA NA NA NA NA 6 NA NA NA NA NA NA 46.0 26.2 0.4 0 0 00 0 NA Not assayed

TABLE 6.b Unselected libraries ELN94 L3.6 0 L3.15 0 L8.15 0

EXAMPLE 5 Identification of Peptide Sequences From Transported Phageacross Caco-2 Tissue Samples

Caco-2 snapwells were prepared as described above and the X30 librarywas screened using Caco-2 cells according to the procedures given above.FIG. 1 summarizes phage yield (% phage transported from the apical tobasolateral medium) at cycles 1, 2, 3 and 4 in the basolateral medium ofpolarized Caco-2 cells grown on snapwells. At each cycle the basolateralmedium was sampled both 1 hour and 24 hour post addition of phage to theapical medium. Thus, following addition of the initial phage library atcycle one, the basolateral medium was removed after one hour andreplaced with fresh basolateral medium. Subsequently, the basolateralmedium was removed 24 hours post addition of the initial phage library.In each case (one hour and 24 hour basolateral medium samples), thephage present was quantitated by titering a sample of each basolateralmedium in Escherichia coli K91Kan strain. The remaining basolateralmedium from the one hour and twenty four hour sampling time point wascombined, the phage present were PEG-precipitated, the precipitatedphage was resuspended in 100 μl of TBS and was used to infectEscherichia coli K91Kan, thus permitting amplification of the phagepresent in the basolateral medium as outlined previously. Followingamplification, the amplified phage was titered, PEG-precipitated,resuspended in TBS and titered. The phage suspension was now ready forthe next round of further selection in the cultured Caco-2 cells, byrepeating the steps above using the phage transported into thebasolateral medium, as outlined previously. Upon going from cycle 1 to4, there was a 19.2 fold enrichment of phage which are transported fromthe apical to basolateral medium of the Caco-2 cells grown on snapwells.

FIG. 2 summarizes the relative binding of 100 different phage isolatesto fixed Caco-2 cells. The 100 individual phages from the X30 librarywere obtained from the cycle 4 selection (transport from apical to thebasolateral medium) of cultured Caco-2 cells grown on snapwells. ForELISA analysis, Caco-2 cells were grown to confluence in 96 well tissueculture plates as described above, followed by fixing in 10%formaldehyde as described above. The ELISA analysis was performed usingthe HRP-anti-M13 conjugate. In this figure, the binding of each phageisolate is arranged or presented so that the “weakest” to “strongest”binding phage are presented from left to right (and not the numericalnumber of the phage isolate). The binding of the negative control phage(M13mp18) and the absorbance readings obtained with untreated fixedCaco-2 cells is shown on the extreme right of FIG. 3, respectively.

FIG. 3 summarizes the binding of the top ten binders, clones 32, 34, 39,40, 53, 80, 84, 97, 98, and 100, to fixed Caco-2 cells, along with thebinding of the negative control phage M13mp18 to the fixed Caco-2 cells,with phage binding monitored by ELISA analysis as described above. Thebinding studies were performed in duplicate, using neat phage (˜10¹⁰pfu/ml) or diluted phage samples (diluted 1:25 and 1:100 in each case).As a control, the absorbance readings obtained using the fixed Caco-2cells in which no phage was added, is shown on the right hand side ofFIG. 3. FIG. 4 is essentially the same as FIG. 3, except that thebackground absorbance readings obtained using the fixed Caco-2 cellsonly, to which no phage was added, has been subtracted from theabsorbance readings obtained using fixed Caco-2 cells which wereincubated with the indicated phage clone samples and the negativecontrol phage M13mp18. The precise titers of neat phage used for eachclone are given in Table 7.

TABLE 7 TITERS OF NEAT PHAGE SAMPLES FOR THE TOP TEN BINDERS CLONEpfu/ml 32 1.19 × 10¹⁰ 34 2.87 × 10¹⁰ 39 1.34 × 10¹⁰ 40 9.09 × 10⁹ 531.89 × 10¹⁰ 80 2.25 × 10¹⁰ 84 1.27 × 10¹⁰ 87 7.99 × 10⁹ 98 1.99 × 10¹⁰100  8.36 × 10⁹

FIG. 5 is a graphical representation of the binding of the phage clones39, 97 and 100, and the negative control phage M13mp18, to fixed Caco-2cells using either neat phage samples (at ˜10¹⁰ pfu/ml) or the samephage diluted 1:25 and 1:100. The phage binding experiments andsubsequent ELISA analysis was performed as previously outlined. Thisdata shows that the phage clones 39, 97 and 100 bind in a dose responsemanner, with reduction in the ELISA absorbance readings obtainedfollowing dilution of the phage either 1:25 or 1:100. In contrast, thenegative control phage M13mp18 does not bind in a dose response manner,with linear absorbance readings obtained using either neat, 1:25 or1:100 diluted phage.

The top ten binders, clones 32, 34, 39, 40, 53, 80, 84, 97, 98 and 100were sequenced using procedures outlined above. Eight of these sequenceswere identical to the sequence of clone 97 giving DNA sequence SEQ. NO.ID: 11 and peptide sequence SEQ. NO. ID: 12. The two remaining clones(53 and 100) produced individual isolates DNA SEQ. NOS. ID: 13 and 15with the corresponding peptide sequences SEQ. NOS. ID: 14 and 16,respectively. One skilled in the art could determine without undueexperimentation which fragments of these peptides permit or facilitatethe transport of an active agnet through a human or animal tissue. Onthe basis of the results of Example 4, it is expected that thesefragments consist of at least 6 amino acid residues.

EXAMPLE 6 Transport of Phage from Rat Lumen into the Portal and SystemicCirculation

In this study, phage from random phage display libraries as well ascontrol phage were injected into the lumen of the rat gastrointestinaltract (in situ rat closed loop model). Blood was collected over timefrom either the systemic circulation or portal circulation and thenumber of phage which were transported to the circulation was determinedby titering blood samples in E. coli. At the conclusion of thecollection of either systemic (300 min) or portal blood (60 min), theanimals were sacrificed and brain, heart, kidney, spleen, ileum,duodenum, liver and pancreas tissue samples were removed, frozen inliquid nitrogen and stored at −80° C.

The phage display libraries used in this study were D38 and DC43 inwhich gene III codes for random 38-mer and 43-mer peptides,respectively. As a negative control, the identical phage M13mp18, inwhich gene III does not code for a “random” peptide sequence, was used.Both the library phages D38 and DC43 were prepared from E. coli, mixedtogether, dialyzed against PBS, precipitated using PEG/NaCl and wereresuspended in PBS buffer. The M13mp18 control was processed in asimilar manner. The titer of each phage sample was determined and thephage samples were diluted in PBS to approximately the same titers priorto injection into the rat closed loop model.

For sampling from the systemic circulation, approximately 15 cm of theduodenum of Wistar rats was tied off (closed loop model), approximately0.5 ml of phage solution was injected into the closed loop and blood(0.4 ml) was sampled from the tail vein at various times. The timepoints used (in min) were: 0, 15, 30, 45, 60, 90, 120, 180, 240 and 300minutes. For sampling from the portal circulation, the portal vein wascatheterized, approximately 15 cm of the duodenum was tied off (closedloop model), 0.5 ml of phage solution was injected into the closed loopand blood was sampled from the portal vein catheter at various times. Asthe portal sampling is delicate, sampling times were restricted to 15,30, 45 and 60 minutes, where possible. The volume of phage injected intoeach animal was as follows:

ANIMALS (15) VOLUME OF PHAGE INJECTED R1-R3 0.50 ml R4 0.43 ml R5-R150.45 mlThe estimated number of transported phage has been adjusted to accountfor differences in volume injected into each animal (using 0.5 ml as thestandard volume).

To investigate transport into the systemic circulation, animals R1, R2and R3 received the control phage M13mp18 and animals R4, R5, R6 and R7received the test phage D38/DC43 mix. To investigate transport into theportal circulation, animals R8, R9 and R10 received the control phageM13mp18 and animals R11, R12, R13 and R14 received the test phageD38/DC43 mix. Animal R15* received the combined phage samples fromanimals R4-R7 (see Table 8) which were sampled from the systemiccirculation on day one, followed by amplifiction in E. coli, PEGprecipitation and resuspension in PBS. On subsequent analysis, the titerof this phage was found to 100 times greater than the other phagesamples used for animals R8-R14. Thus, the date presented for animal R15in Table 9 is adjusted down.

Approximately 0.4 ml of the blood was collected at each time point ineach model system. 30 μl of the collected blood (systemic) was mixedwith 100 μl of the prepared E. coli strain K91Kan, incubated at 37° C.for 30 min, and plated out for plaque formation using Top Agarose on LBplates. Various negative controls were included in the titeringexperiments. The following day the number of plaques forming units(pfu's) was determined. Similarly, 30 μl of the collected blood (portal)and serial dilutions (1:100, 1:1000) thereof was mixed with 100 μl ofthe prepared E. coli strain K91Kan, incubated at 37° C. for 30 min, andplated out for plaque formation using Top Agarose on LB plates. Thefollowing day the number of plaques forming units (pfu's) wasdetermined.

In addition, approximately 300 μl of the collected blood from each timepoint (systemic and portal) was incubated with 5 ml of prepared E. colistrain K91Kan in modified growth media containing 5 mM MgCl₂/MgSO4,incubated at 37° C. overnight with shaking (to permit phageamplification). The samples were centrifuged and the cell pellet wasdiscarded. Samples of the phage supernatant were collected seriallydiluted (10⁻², 10⁻⁴, 10⁻⁶, 10⁻⁸) in TBS buffer and were plated forplaques in order to determine the number of pfu's present in theamplified phage samples.

Furthermore, an aliquot of phage was removed from the “amplified”supernatants obtained from test animals #R4-R7 (samples from each timepoint were used), combined and was PEG-precipitated for two hours. Theprecipitated phage was resuspended in PBS buffer and was injected intoclosed loop model of animal #R15, followed by portal sampling.

The number of phage transported from the closed loop model into thesystemic circulation is presented in Table 8. The number of phagetransported from the closed loop model into the portal circulation ispresented in Table 9. These numbers are corrected for phage inputdifference and for volume input differences. Clearly, more phage arepresent in the portal samples than in the systemic samples, indicativeof either hepatic or RES clearance and/or phage instability in thesystemic circulation. In addition, the uptake of phage from the GIT intothe portal circulation is quite rapid, with substantial number of phagesdetected within 15 minutes. The results from the portal samplingexperiments would also indicate that the kinetics of uptake of phagefrom the D38/DC43 libraries is quicker than that of the control phage.Thus, there may be preferential uptake of phage coding for randompeptide sequences from the GIT into the portal circulation. In the caseof animals R13, R14 and R15*, the % of the phage transported into thetitered blood sample within the limited time frame (30, 45 and 15 mins,respectively) is estimated as 0.13%, 1.1% and 0.013%, respectively.

TABLE 8 NUMBER OF PHAGE TRANSPORTED FROM THE CLOSED LOOP MODEL INTO THESYSTEMIC CIRCULATION Time (min) R1 R2 R3 R4 R5 R6 R7  0 0 0 0 0 0 0 0 15 0 1 9 0 0 1 7  30 2 1 0 0 46 1 11  45 10 4 2 1 32 0 20  60 63 19 211 114 0 21  90 104 20 18 3 115 0 22 120 94 24 27 0 64 0 6 180 94 12 23 1413 0 0 240 14 1 20 0 36 0 0 300 1 1 4 2 0 0 0 Total number of 382 83124 8 820 2 87 transported phage Animals R1, R2 and R3 received thecontrol phage M13mp18 Animals R4, R5, R6 and R7 received the test phageD38/DC43 mix

TABLE 9 NUMBER OF PHAGE TRANSPORTED FROM THE CLOSED LOOP MODEL INTO THEPORTALCIRCULATION Time (min) R8 R9 R10 R11 R12 R13 R14 R15* 15 15 6 3 119 231,000 1,000,000 20,000 30  1 5 26 — 0 60,000 272,000 — 45 — 1 555 —1 — 1,240,000 — 60 — — — — 420,000 — — — Animals R8, R9 and R10 receivedthe control phage M13mp18 Animals R11, R12, R13 and R14 received thetest phage D38/DC43 mix Animal R15* received the combined phage samplesfrom animals R4-R7 (see Table 8) which were sampled from the systemiccirculation on day one, followed by PEG precipitation and resuspensionin PBS. On subsequent analysis, the titer of this phage was found to be100 times greater than the other phage samples used for animals R8-R14.Thus, the date presented for animal R15* in Table 9 is adjusted down.

These studies demonstrate that both the control phage and the D38/DC43phages are transported over time from the lumen of the GIT into theportal and systemic circulation, as demonstrated by titering the phagetransported to the blood in E. coli. More phage are transported from thetest phage samples into the portal circulation than the correspondingcontrol phage sample. In addition, the kinetics of transport of the testphage into the portal circulation does appear to exceed that of the.control phage. Phage from the D38/DC43 libraries which appeared in thesystemic circulation of different animals (R4-R7) were pooled, amplifiedin E. coli, precipitated, and re-applied to the lumen of the GIT,followed by collection in the portal circulation and titering in E.coli. These selected phage were also transported from the lumen of theGIT into the portal circulation. This in situ loop model may representan attractive screening model in which to identify peptide sequenceswhich facilitate transport of phage and particles from the GIT into thecirculation.

The frozen brain tissues for all the animals were thawed, cut into smallpieces, resuspended in 5 ml of sterile PBS containing a cocktail ofprotease inhibitors (Boehringer) and homogenized in an Ultrathorexhomogenizer. Serial dilutions (neat and 10⁻², 10⁻⁴, 10⁻⁶ dilutions) weretitered in E. coli. K91Kan starved bacteria. In addition, aliquots (100μl) of the tissue homogenate were added to 100 μl of E. coli K91Kanstarved bacteria, incubated at 37° C. for 10 min, followed by additionof 5 ml of LB medium and incubation overnight at 37° C. in a rotatingincubator. Serial dilutions of amplified phage were then titered in E.coli. No phage was detected in tissue homogenate samples obtained fromanimals R4-R7 (sacrificed after systemic sampling) prior toamplification but phage were detected after amplification. Phage weredetected prior to amplification in tissue homogenate samples obtainedfrom animals R11-R14.

An equal amount of phage from animals R11 through R14 were pooled at aconcentration of 5×10¹¹ pfu/ml (in PBS). These phage were amplified asabove, plated and the recombinant phage were picked and purified.Sequencing templates were prepared using the QIAprep Spin M13 columnsand were sequenced using the primer SEQ. NO. ID: 17 and SequenaseVersion 2.0 DNA sequencing kit. Eight DNA sequences (SEQ. NOS. ID: 18,20, 22, 24, 26, 28, 30, 32) with corresponding peptide sequences SEQ.NOS. ID: 19, 21, 23, 25, 27, 29, 31 and 33 were discovered. Thesepeptide sequences, which were isolated because of their ability totransport phage (particles) from the GI tract to the brain, are capableof facilitating the transport of an active agent, such as a micro- ornanoencapsulated active agent, through a human or animal tissue.Additionally, one skilled in the art could determine without undueexperimentation which fragments or analogs of these peptides permit orfacilitate the transport of an active agnet through a human or animaltissue. On the basis of the results of Example 4, it is expected thatthese fragments consist of at least 6 amino acids.

Using this screening model system, a number of preselected phagelibraries now exist. These are the one pass brain tissue phage library,the one pass systemic phage library from animals R4-R7, a one-passportal library from animals R11-R14 and the two pass, rapid transport,systemic-portal phage library SP-2 from animal R15*.

EXAMPLE 7 Transport of Phage from Preselected Phage Libraries from theRat Lumen into the Portal and Systemic Circulation

Four preselected phage libraries, GI-D, GI-S, GI-H and GI-P, areconstructed by pooling phage previously selected by screening randomphage display libraries D38 and DC43 using four distinct receptor orbinding sites located in the GIT. Similar to Example 7 above, thesepreselected phage libraries together with the negative control phageM13mp18 are injected into the rat closed loop model (6 animals perpreselected phage library), blood is collected over time from the portalcirculation via the portal vein and, at the termination of theexperiment, a systemic blood sample is collected from the tail vein andthe intestinal tissue region from the closed loop is collected.

In particular, phages selected in vitro to each receptor or binding sitelocated in the GIT were amplified in E. coli, PEG-precipitated,resuspended in TBS and the titer of each phage sample was determined byplaquing in E. coli as described above. Subsequently, an equal number ofeach phage (8×10⁸ phage) for each receptor site was pooled into apreselected phage library together with the negative control phageM13mp18 and each preselected phage library was administered to 6 Wistarrats per library (rats 1-6; GI-D, rats 7-12; GI-S, rats 13-18; GI-P, andrats 19-24; GI-H). Using the in situ loop model described above, 0.5 mlof preselected phage library solution was injected into the tied-offportion of the duodenum/jejunum. Blood was collected into heparinisedtubes from the portal vein at 0, 15, 30, 45 and 60 minutes. A bloodsample was taken from systemic circulation at the end of the experiment.Similarly, the portion of the duodenum/jejunum used for phage injectionwas taken at the end of the experiment.

30 μl of the collected portal blood (neat and 10⁻², 10⁻⁴, 10⁻⁶dilutions) was added to 30 μl E. coli K91Kan cells (overnight cultureand incubated at 37° C. for 10 min. Subsequently, 3 ml of top agarosewas added and the samples were plated for plaques. 100 μl of thecollected portal blood was added to 100 μl of E. coli K91Kan. 5 ml of LBmedium was then added and the samples were incubated at 37° C. overnightin a rotating microbial incubator. The E. coli was removed bycentrifugation and the amplified phage supernatant samples were eithertitered directly or were PEG-precipitated, resuspended in TBS andtitered. Following titration of the amplified phage, samples containingphage from each set of animals were combined, adjusting the titer ofeach sample to the same titer, and were plated for plaques on LB agarplates (22 cm² square plates). Either 12,000 or 24,000 phage were platedfor plaques.

30 μl of the collected systemic blood (neat and 10⁻², 10⁻⁴, 10⁻⁶dilutions) was added to E. coli K91Kan cells, incubated at 37° C. for 10min. Three ml of top agarose was then added and the samples were platedfor plaques. 100 μl of the collected systemic blood was added to 100 μlof E. coli K91Kan, incubated at 37° C. for 10 min. 5 ml of LB medium wasthen added and the samples were incubated at 37° C. overnight in arotating microbial incubator. The E. coli was removed by centrifugationand the amplified phage supernatant samples were either titered directlyor were PEG-precipitated, resuspended in TBS and titered. Followingtitration of the amplified phage, samples containing phage from each setof animals were combined, adjusting the titer of each sample to the sametiter, and were plated for plaques on LB agar plates (22 cm² squareplates). Either 12,000 or 24,000 phage were plated for plaques.

The intestinal tissue portion used in each closed loop was excised. Thetissue was cut into small segments, followed by 3 washings in sterilePBS containing protease inhibitors, and homogenized in an Ultra thorexhomogeniser (Int-D samples). Alternatively, the tissue (in PBSsupplemented with protease inhibitors) was homogenized in an UltraThorex homogeniser, washed 3 times in PBS containing protease inhibitorsand resuspended in PBS containing protease inhibitors (Int-G samples).In each case, serial dilutions (neat and 10⁻², 10⁻⁴, 10⁻⁶ dilutions) ofthe tissue homogenate was titered in E. coli. In addition, an aliquot(100 μl) of the tissue homogenate was added to 100 μl of E. coli K91Kan,incubated at 37° C. for 10 min. followed by addition of 5 ml of LBmedium and incubation overnight at 37° C. in a rotating microbialincubator.

The phage amplified from the portal blood, systemic blood and intestinaltissue was plated for plaques. The plaques were transferred to Hybond-NNylon filters, followed by denaturation (1.5M NaCl, 0.5M NaOH),neutralization (0.5M TRIS-HCl, pH 7.4, 1.5M NaCl), washing in 2×SSCbuffer. The filters were air-dried, and the DNA was cross-linked to thefilter (UV crosslinking: 2 min, high setting). The filters wereincubated in pre-hybridization buffer (6×SSC, 5×Denhardt's solution,0.1% SDS, 20 μg/ml yeast tRNA) at 40° C.-45° C. for at least 60 min.

Synthetic oligonucleotides, (22-mers), complimentary to regions codingfor the receptor or binding sites used to create the preselected phagelibrary, were synthesized. The oligonucleotides (5 pmol) were 5′ endlabelled with ³²P-ATP and T4 polynucleotide kinase and approximately 2.5pmol of labelled oligonucleotide was used in hybridization studies.Hybridization's were performed at 40-45° C. overnight in buffercontaining 6×SSC, 5×Denhardt's solution, 0.1% SDS, 20 μg/ml yeast tRNAand the radiolabeled synthetic oligonucleotide, followed by washings(20-30 min at 40-45° C.) in the following buffers: (i) 2×SSC 0.1% SDS,(ii) 1×SSC/0.1% SDS, (iii) 0.1×SSC/0.1% SDS. The filters were air-driedand exposed for autoradiography for 15 hours, 24 hours or 72 hours.

Table 10 summarises the results from the hybridization studies outlinedabove. Apart from the synthetic oligonucleotide to HAX9, alloligonucleotides were initially confirmed to be radiolabeled, asdetermined by hybridisation to the corresponding phage target (eg.,phage S15 hybridised to the oligonucleotide S15). In addition, under theexperimental conditions used the oligonucleotides essentially did nothybridise to the negative control phage template M13mp18. Twooligonucleotides were synthesised to the phage M13mp18—(1) a positiveoligonucleotide which hybridises to a conserved sequence in both M13mp18and each of the GIT receptor or GIT binding site selected phages[designated M13(positive)] in Table 10 and (2) a negativeoligonucleotide which only hybridises to a sequence unique to themultiple cloning site of phage M13mp18 and which does not hybridise toany of the GIT receptor or GIT binding site selected phages.

TABLE 10 SUMMARY OF HYBRIDIZATION RESULTS A: (GI-S) Phage Portal Int.-GInt.-D S15 ++ +/− +/− S21 − − − S22 − −/+ − SNI-10 +++/+ ++ ++ SNI-28 −− − SNI-34 ++ − − SNI-38 ++ − − SNI-45 − − − SNIAX-2 − − − SNIAX-6- − −SNIAX-8 − − − M13 (positive) ++++++ ++++++ ++++++ M13 (negative) ND + −B: (GI-D) Phage Portal Int.-G Int.-D DAB3 +++ +/− −/+ DAB7 ++ ++ −/+DAB10 ++++++ +/− −/+ DAB18 − − − DAB24 − − − DAB30 ++++ ++ +++ DAX15 − −− DAX23 −/+ + −/+ DAX24 − − − DAX27 − + − DCX8 +++++ +/− − DCX11 ++++++++ −/+ DCX26 − − − DCX33 +++ ++ ++ DCX36 − − − DCX39 − −/+ − DCX42 − −−/+ DCX45 − ++ − M13 (positive) ++++++ ++++++ ++++++ M13 (negative) +/−−/+ − C: (GI-H) Phage Int-G Portal Systemic H40 − − ++++ HAX9 ND ND NDHAX35 − + − HAX40 − − − HAX42 − ++ ++ HCA3 − − − PAX2 − +++ ++++ M13(positive) ++++++ ++++++ ++++++ M13 (negative) − −−/+ − D: (GI-P) PhageInt-G Portal Systemic PAX2 − ++ − PAX9 ++ +++ − PAX14 − ++ − PAX15 −/+ −− PAX16 − − − PAX17 + ++/+ − PAX18 − − − PAX35 − − − PAX38 −/+ − −PAX40 + +++ − PAX43 + − − PAX45 − − − PAX46 − +++ − P31 ++ ++++ ++ P90 −− − 5PAX3 ++/+ ++ − 5PAX5 − − ++ 5PAX7 +++ − − 5PAX12 ++++ ++ − H40 ++++ − M13 (positive) ++++++ ++++++ ++++++ M13 (negative) − − −

In the case of the GI-S pool of phages, only four phages are transportedfrom the closed loop model into the portal circulation—phages S15,SNI-10, SNI-34 and SNI-38. The other phages, S21, S22, SNI-28, SNI45,SNIAX-2, SNIAX-6 and SNIAX-8 are not transported from the GIT into theportal circulation. In addition, phages SNI-10 and to a lesser extentphages S15 and S22 were found in the intestine samples or fractions,whereas the other phages were not. There was a very low presence (<0.1%)of the phage M13mp18 in the Int-G samples. These results show thatphages can be further selected from pre-selected libraries, permittingthe identification of phages which are transported from the GIT closedloop into the portal circulation or phages which bind to or areinternalised by intestinal tissue.

In the case of the GI-D pool of phages, there is a rank order by whichphages are transported from the GIT closed loop model into the portalcirculation, with phages DCX11 and DAB 10 preferably transported,followed by phages DCX8, DAB30, DAB3 and DAB7. A number of phages fromthis pool are not transported into the portal circulation, includingphages DAB18, DAB24, DAX15, DAX24, DAX27, DCX26, DCX36, DCX39, DCX42,DCX45. There is a very low level of transport of phage DAX23 from theGIT into the portal circulation. Similarly, only some of the phages arefound in the intestinal samples fractions, including phages DAB30,DCX33, DAB7, DCX11, DCX45 and to a much lesser extent phages DAB3,DAB10, DCX8, DCX39, DCX42. Some phages are not found in the intestinalsamples, including phages DAB18, DAB24, DAX15, DAX24, DCX26, and DCX36.There was a very low presence (<0.1%) of the phage M13mp18 in the Int-Gsamples. These results show that phages can be further selected frompre-selected libraries, permitting the identification of phages whichare transported from the GIT closed loop into the portal circulation orphages which bind to or are internalised by intestinal tissue.

In the case of the GI-H pool of phages, there is a rank order by whichphages are transported from the GIT closed loop model into the portal orsystemic circulation, with phages PAX2 (which was used at a 4×concentration relative to the other phages in this pool) followed byphage HAX42 found in the portal and systemic circulation and phage H40found in the systemic circulation only. None of the phages in this poolwere found in the intestine samples or fractions. Phage M13mp18 was notfound in the intestine fractions or systemic circulation, with very lowincidence (<0.001%) in the portal circulation. These results show thatphages can be further selected from pre-selected libraries, permittingthe identification of phages which are transported from the GIT closedloop into the portal and/or systemic circulation or phages which bind toor are internalised by intestinal tissue.

In the case of the GI-P pool of phages, the phages PAX2 and H40 werealso included in this pool. A number of phages from this pool were foundin the portal circulation, including phages P31, PAX46, PAX9, H40,PAX17, PAX40, PAX2, PAX14, 5PAX3 and 5PAX12. A number of phages were notfound in the portal blood including the negative control phage M13mp18,PAX15, PAX16, PAX18, PAX35, PAX38, PAX43, PAX45, P90, 5PAX5 and 5PAX7.The only phage found in the systemic circulation were phages 5PAX5 andP31. In addition, there was preferential binding of some phages to theintestine, including phages 5PAX12, 5PAX7, 5PAX3, H40, P31, PAX9, and toa lesser extent phages PAX38 and PAX5. Some phages were not found in theintestine samples, including the negative control phage M13mp18 and thephages PAX2, PAX14, PAX16, PAX18, PAX35, PAX45, PAX46, P90 and 5PAX5.These results show that phages can be further selected from pre-selectedlibraries, permitting the identification of phages which are transportedfrom the GIT closed loop into the portal and/or systemic circulation orphages which bind to or are internalised by intestinal tissue.

The present invention is not to be limited in scope by the specificembodiments described herein. Various modifications of the invention inaddition to those described herein will become apparent to those skilledin the art from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

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